A vacuolar H(+)-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress.

BACKGROUND: Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.). METHODS: The vacuolar H(+) pumps, the H(+)-ATPase and H(+)-PPiase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops. RESULTS: In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H(+) transport, which was in turn at least 3-folds faster than that mediated by the H(+)-PPiase. Its PPi hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PPi-dependent H(+)-gradient. This effect was antagonized by plant rehydration as the H(+)-PPiase activity was highly stimulated, reassuming a coupled PPi-driven H(+) pumping. Maize tonoplasts exhibited 2-4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PPi-dependent H(+) pumping in response to stress relief, resulting in an enhanced H(+)-pumps coupling efficiency. CONCLUSION: These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H(+)-pumps, which differ between weeds and crops upon water deficit. GENERAL SIGNIFICANCE: The evidences highlight an unusual modulation of the H(+)-PPiase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.

Bioinspired peptides induce different cell death mechanisms against opportunistic yeasts.

The management of fungal diseases imposes an urgent need for the development of effective antifungal drugs. Among new drug candidates are the antimicrobial peptides, and especially their derivatives. Here, we investigated the molecular mechanism of action of three bioinspired peptides against the opportunistic yeasts Candida tropicalis and Candida albicans. We assessed morphological changes, mitochondrial functionality, chromatin condensation, ROS production, activation of metacaspases, and the occurrence of cell death. Our results indicated that the peptides induced sharply contrasting death kinetics, of 6 h for RR and 3 h for D-RR to C. tropicalis and 1 h for WR to C. albicans. Both peptide-treated yeasts exhibited increased ROS levels, mitochondrial hyperpolarization, cell size reduction, and chromatin condensation. RR and WR induced necrosis in C. tropicalis and C. albicans, but not D-RR in C. tropicalis. The antioxidant ascorbic acid reverted the toxic effect of RR and D-RR, but not WR, suggesting that instead of ROS there is a second signal triggered that leads to yeast death. Our data suggest that RR induced a regulated accidental cell death in C. tropicalis, D-RR induced a programmed cell death metacaspase-independent in C. tropicalis, while WR induced an accidental cell death in C. albicans. Our results were obtained with the LD100 and within the time that the peptides induce the yeast death. Within this temporal frame, our results allow us to gain clarity on the events triggered by the peptide-cell interaction and their temporal order, providing a better understanding of the death process induced by them.

Multi-cancer V-ATPase molecular signatures: A distinctive balance of subunit C isoforms in esophageal carcinoma.

BACKGROUND: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. METHODS: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. FINDINGS: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. INTERPRETATION: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms. FUNDING: This work was supported by grants from CNPq and FAPERJ-Brazil.

Myrtenal-induced V-ATPase inhibition - A toxicity mechanism behind tumor cell death and suppressed migration and invasion in melanoma.

BACKGROUND: Metastatic tumor cells have acidic extracellular pH and differential electrochemical H+ gradients generated across their cell membranes by V-type H+-ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice. METHODS: The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach. RESULTS: The inhibition of V-ATPases by 100 µM Myrtenal disrupted the electrochemical H+ gradient across the cell membranes, strongly induced cell death (4-5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15 mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers. CONCLUSIONS: These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H+ fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition. GENERAL SIGNIFICANCE: The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential.

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H(+) gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H(+)-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H(+)-ATPase (V-ATPase) even in the absence of the H(+) gradient in a water-Me2SO co-solvent mixture, resulting in net synthesis of [γ-(32)P]ATP from [(32)P]Pi and ADP. The water-Me2SO (5 to 20 %) media promoted inhibition of both PPi hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH4 (+)). This effect of Me2SO on the ATP⇔(32)P exchange reaction seems to be related to a decrease of the apparent K m of the V-ATPase for Pi. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.

Morpho-pathological and ultrastructural analysis of the murinemelanoma B16F10 and B16F0 cell lines and derived tumors / Anáalise morfo-patolóogica e ultraestrutural de linhagens celulares B16F10 E B16F0 e melanoma decamundongo e tumores derivados

Murine B16 cell lines of tumorigenic melanocytes have been widely used as cellular model for biochemicaland molecular studies in melanoma research. Despite such importance there is still a lack of information onthe microanatomy and cytopathology of these B16 models. The aim of this study was to shed light on themorpho-pathological specificities of the murine melanoma cell lines B16F10 and B16F0, and derived tumors,and correlate the findings with distinct metastatic potential. Cyto- and histopathological analysis carried outby optical and transmission electron microscopy (TEM) revealed that both tumor cell lines exhibit typicalfeatures of carcinogenic cells such as atypical mitoses, multiple nucleoli and enlarged nucleus. In agreementwith their highest metastatic potential, B16F10 cells and tumors exhibited more aberrant morphology thanthat observed in B16F0 samples. Also the histopathological analysis revealed that only the B16F10 tumor cellscould invade adjacent muscle tissues. Under TEM, B16F0 cell line presented mitochondrial hyperplasia andhypertrophia and a large number of melanosomes, while B16F10 showed a larger number of mitochondria ina wider range of shapes and sizes, numerous polyribosomes, cellular peripheral fibrillar material, and abnormalclusters of mitochondria. These morphological features related to the differential metastatic potentials ofB16F10 and B16F0, provide new insights on the cellular changes involved in melanoma metastatic progressionand also would be instrumental to expand the application of these cell lines as models in melanoma research.

Linhas de células B16 de melanócitos tumorais de camundongos têm sido amplamente utilizadascomo modelo celular para estudos bioquímicos e moleculares na pesquisa de melanoma. Apesardessa importância, ainda há uma falta de informação sobre a micro anatomia e citopatologia destesmodelos B16. O objetivo deste estudo foi pesquisar sobre as especificidades morfo-patológicas das linhasde células de melanoma de camundongos B16F10 e B16F0 e tumores derivados, e correlacionaros achados com potencial metastático distinto. Cito e análise histopatológica realizada por microscopiaóptica e eletrônica de transmissão (TEM) revelou que ambas as linhagens celulares tumoraisexibem características típicas de células cancerígenas, como mitoses atípicas, múltiplos nucléolos enúcleo aumentado. De acordo com seu potencial mais elevado, as células B16F10 e tumores metastáticosexibiram morfologia mais aberrante do que a observada em amostras de B16F0. A análisehistopatológica também revelou que apenas as células tumorais B16F10 poderiam invadir os tecidosmusculares adjacentes. De acordo com TEM, a linhagem de células B16F0 apresentou hiperplasiae hipertrofia mitocondrial e um grande número de melanossomas, enquanto B16F10 demonstrouum maior número de mitocôndrias em uma ampla gama de formas e tamanhos, numerosos polirribossomas,material fibrilar celular periférico e aglomerados de mitocôndrias anormais. Estascaracterísticas morfológicas relacionadas aos diferenciais pote nciais metastáticos de B16F10 e B16F0forneceram novas compreensões sobre as mudanças celulares envolvidas na progressão metastáticade melanoma e também seria fundamental para ampliar a aplicação dessas linhagens celulares comomodelo na pesquisa melanoma.

Morpho-pathological and ultrastructural analysis of the murinemelanoma B16F10 and B16F0 cell lines and derived tumors / Anáalise morfo-patolóogica e ultraestrutural de linhagens celulares B16F10 E B16F0 e melanoma decamundongo e tumores derivados

Murine B16 cell lines of tumorigenic melanocytes have been widely used as cellular model for biochemicaland molecular studies in melanoma research. Despite such importance there is still a lack of information onthe microanatomy and cytopathology of these B16 models. The aim of this study was to shed light on themorpho-pathological specificities of the murine melanoma cell lines B16F10 and B16F0, and derived tumors,and correlate the findings with distinct metastatic potential. Cyto- and histopathological analysis carried outby optical and transmission electron microscopy (TEM) revealed that both tumor cell lines exhibit typicalfeatures of carcinogenic cells such as atypical mitoses, multiple nucleoli and enlarged nucleus. In agreementwith their highest metastatic potential, B16F10 cells and tumors exhibited more aberrant morphology thanthat observed in B16F0 samples. Also the histopathological analysis revealed that only the B16F10 tumor cellscould invade adjacent muscle tissues. Under TEM, B16F0 cell line presented mitochondrial hyperplasia andhypertrophia and a large number of melanosomes, while B16F10 showed a larger number of mitochondria ina wider range of shapes and sizes, numerous polyribosomes, cellular peripheral fibrillar material, and abnormalclusters of mitochondria. These morphological features related to the differential metastatic potentials ofB16F10 and B16F0, provide new insights on the cellular changes involved in melanoma metastatic progressionand also would be instrumental to expand the application of these cell lines as models in melanoma research.AU

Linhas de células B16 de melanócitos tumorais de camundongos têm sido amplamente utilizadascomo modelo celular para estudos bioquímicos e moleculares na pesquisa de melanoma. Apesardessa importância, ainda há uma falta de informação sobre a micro anatomia e citopatologia destesmodelos B16. O objetivo deste estudo foi pesquisar sobre as especificidades morfo-patológicas das linhasde células de melanoma de camundongos B16F10 e B16F0 e tumores derivados, e correlacionaros achados com potencial metastático distinto. Cito e análise histopatológica realizada por microscopiaóptica e eletrônica de transmissão (TEM) revelou que ambas as linhagens celulares tumoraisexibem características típicas de células cancerígenas, como mitoses atípicas, múltiplos nucléolos enúcleo aumentado. De acordo com seu potencial mais elevado, as células B16F10 e tumores metastáticosexibiram morfologia mais aberrante do que a observada em amostras de B16F0. A análisehistopatológica também revelou que apenas as células tumorais B16F10 poderiam invadir os tecidosmusculares adjacentes. De acordo com TEM, a linhagem de células B16F0 apresentou hiperplasiae hipertrofia mitocondrial e um grande número de melanossomas, enquanto B16F10 demonstrouum maior número de mitocôndrias em uma ampla gama de formas e tamanhos, numerosos polirribossomas,material fibrilar celular periférico e aglomerados de mitocôndrias anormais. Estascaracterísticas morfológicas relacionadas aos diferenciais pote nciais metastáticos de B16F10 e B16F0forneceram novas compreensões sobre as mudanças celulares envolvidas na progressão metastáticade melanoma e também seria fundamental para ampliar a aplicação dessas linhagens celulares comomodelo na pesquisa melanoma.(AU)